Single tube confirmation PCR protocol

The following colony PCR protocol has been designed to be performed in individual
reaction tubes. We usually test three colonies from each transformation along
with a wild-type control. A series of five different PCR tests are performed
on each colony to confirm both of the novel recombination junctions .

1. Clonal purification

- Pick three colonies from a transformation plate and streak each one out
to single colonies on separate G418 containing plates (200mg/l).

- Repeat this step for each of the 3 isolates and the wild-type control.

- Incubate the Zymolyase solutions at 37 ° C for 30 minutes followed by
10 minutes at 95 ° C.

*It is not necessary to remove the Zymolyase solution by pelleting the cells
before the PCR.

*The Zymolyase treated cells can be stored at 4 ° C for several days before
being used as template for the colony PCR. However, fresher is probably better.

*The amount of cells that gets picked into the Zymolyase does
not seem to be too critical - similar PCR results were obtained when 1 µl,
5 µl or 10 µl of the Zymo treated cells were used as template.

3. PCR reactions

- The lyophilized confirmation primers (~5-10 nmoles of each oligonuleotide)
should be resuspended in 750 µl of TE (final concentration of ~10 µM).
We use 5 µl of each primer in 50 µl PCR reactions (~ 1 µM
final primer concentration).

- Label 20 thin-wall PCR tubes (5 for each isolate) and add the following
primer pairs: A-B, A-kanB, C-D, kanC-D, and A-D. Tubes 1-5 are for isolate
#1, 6-10 are for isolate #2, ..., and tubes 16-20 are for the wild-type control.
Each of the 20 tubes should contain 10 µl of the primer mix (5 µl
of each primer).

- Add the 5 µl of the Zymo treated cells to each of the 20 PCR reactions.
For example, add 5 µl of the Zymo solution from isolate #1 to PCR tubes
1-5, 5 µl of the Zymo solution from isolate #2 to PCR tubes 6-10 and
so on.

*The Taq Polymerase should be added last and PCR mixture should be kept on
ice until the PCR is started. Alternatively, a "hot start" can be
performed by adding the Taq polymerase to the individual tubes after the PCR
mixtures have been heated to 94°C. Hot start improves the PCR results
but this step is labor intensive and we have not found it to be necessary.